Recently, as studies on the structures and functions of sugar chains are rapidly progressing, the development of the use of physiologically active oligosaccharides, glycolipids and glycoproteins and the like as drugs or functional materials has received attention. Among them, sialic acid-containing sugar chains having N-acetylneuraminic acid (NeuAc) at the terminal end have important functions, such as functioning as receptors upon cell adhesion or virus infection.
The sialic acid-containing sugar chains are generally synthesized through the catalysis of sialic acid transferase. Sialic acid transferase is an enzyme that uses CMP-N-acetylneuraminic acid (CMP-NeuAc) as a sugar donor to transfer sialic acid to receptors such as sugar chains. However, CMP-NeuAc that is used as a sugar donor is very expensive and is supplied only in a small amount only enough to be used for a reagent.
Known methods for producing CMP-NeuAc include a method for synthesizing CMP-NeuAc by the action of CMP-NeuAc synthetase using 5′-cytidine triphosphate (CTP) and N-acetylneuraminic acid (NeuAc) as substrates, but CTP and NeuAc that are used as raw materials for producing CMP-NeuAc are expensive, and thus CMP-NeuAc synthesized using these materials is also expensive.
Recently, a method for combining Brevibacterium ammoniagenes, which converts orotic acid to uridine 5′-triphosphate (UTP), with a recombinant E. coli, which produces CTP synthetase catalyzing a reaction for converting UTP to CTP, and a recombinant E. coli, which produces CMP-NeuAc synthetase, and synthesizing CMP-NeuAc in the recombinant organisms using orotic acid and NeuAc as raw materials, was developed. Although this method does not use expensive CTP, it is not considered to be a practical method, because the process is complicated, for example, due to the preparation of a recombinant strain using a plurality of kinds of bacterial strains, a large-sized system for carrying out the process should be prepared, and expensive NeuAc is used as a raw material.
Meanwhile, with respect to the preparation of NeuAc, a method comprising collecting colominic acid, which is a sialic acid polymer, from microorganisms, and chemically decomposing the collected colominic acid, is known, but a method for producing NeuAc using an enzyme was recently developed.
Reported methods for producing CMP-neuraminic acid (CMP-NeuAc) include: (1) a method for producing CMP-NeuAc from N-acetyl-D-mannosamine (ManNAc) using N-acetylneuraminic acid lyase or N-acetylneuraminic acid synthetase (J. Am. Chem. Soc., 110:6481, 1988; J. Am. Chem. Soc., 110:7159, 1988; Japanese Patent Publication 10-4961); (2) a method for producing CMP-NeuAc by converting N-acetylglucosamine (GlcNAc) to N-acetylmanosamine (ManNAc) in an alkaline condition and adding N-acetylneuraminic acid lyase or N-acetylneuraminic acid synthetase thereto to produce N-acetylneuraminic acid (NeuAc) (Japanese Patent Pubblication 5-211884; Biotechnol. Bioeng., 66:2, 1999; Enzyme Microb. Technol., 20, 1997), (3) a method for producing CMP-NeuAc from GlcNAc using N-acetylglucosamine (GlcNAc) 2-epimerase catalyzing the conversion of GlcNAc to MamNAc, and NeuAc lyase or synthetase (WO 95/26399; Japanese Patent Publication 3-180190; Japanese Patent Publication 2001-136982); and (4) a method for synthesizing CMP-N-acetylneuraminic acid using E. coli and yeast strains (WO 2004/009830).
However, the method (1) has a problem in that the raw material N-acetylmanosamine (ManNAc) is expensive, and the method (2) uses inexpensive N-acetylglucosamine (GlcNAc), but has a problem in that the process of purifying ManNAc in the mixture of GlcNAc and N-acetylmanosamine (ManNAc) is complicated. Also, the method (3) has a problem in that, because GlcNAc2-epimerase requires expensive ATP, ATP must be added or must be produced from ATP precursor adenine using microorganisms, and the method (4) has a problem in that the process is complicated due to the use of E. coli and yeast cells.
N-acetylglucosamine-2-epimerase is known to be found in the kidneys, liver, spleen, brain, intestinal mucosa, thymus, pancreas and salivary gland of pigs or rats, the characteristics thereof have been examined with respect to pig-derived enzymes, and the epimerase enzyme has been extracted mainly from animal-derived genes and studied (Biochemistry, 17:3363, 1970; Biochem. J., 210:21, 1983; PNAS, 52:371, 1964). Also, as the N-acetylglucosamine-2-epimerase gene, a pig-derived gene is known (J. Biol. Chem., 271:16294, 1996). Among microorganisms, the entire genome sequence of Synechocystis sp. PCC 6803), a kind of Cyanobacteria, was determined, and N-acetylglucosamine-2-epimerase was identified from the genome (DNA Research, 3:109, 1996; Nucleic Acids Research, 26:63, 1998).
Meanwhile, Korean Patent Publication 10-2001-0102019 relates to N-acetylglucosamine-2-epimerase and DNA encoding the enzyme and discloses the use of DNA encoding N-acetylglucosamine-2-epimerase, which the inventors found in Synechocystis, a kind of Cyanobacteria. 
Also, Korean Patent Publication 10-2006-0010706 discloses a method for producing CMP-N-acetylneuraminic acid either by adding cytidine monophosphate (CMP), N-acetylglucosamine, pyruvic acid and yeast to a recombinant strain transformed with a co-expression vector containing a gene encoding N-acetylglucosamine-2-epimerase and a gene encoding N-acetylneuraminic acid aldolase, thus producing neuraminic acid, and then adding CMP-N-acetylneuraminic acid synthetase to the produced neuraminic acid, or by adding cytidine monophosphate (CMP), N-acetylglucosamine, pyruvic acid and yeast to a recombinant strain transformed with a co-expression vector containing a gene encoding N-acetylneuraminic acid aldolase and a gene encoding CMP-N-acetylneuraminic acid synthetase. However, the method has problems in that several steps should be carried out to produce CMP-N-acetylneuraminic acid and in that cytidine monophosphate (CMP) that is used as a substrate is converted into cytidine triphosphate (CTP) in low yield.
Accordingly, the present inventors have made extensive efforts to develop a method for producing CMP-N-acetylneuraminic acid in an economical and simple manner. As a result, the present inventors have found a novel N-acetylglucosamine-2-epimerase enzyme in Bacteroides fragilis NCTC 9343 and confirmed that CMP-N-acetylneuraminic acid can be produced in high yield through a one-pot reaction using a mixture of various substrates and enzymes, including N-acetylglucosamine-2-epimerase, obtained by transforming the gene for said enzyme with a recombinant vector, inexpensive substrate cytidine monophosphate (CMP) and a very small amount of NTP, thereby completing the present invention.